CN114790194B

CN114790194B - Fibroblast activation protein inhibitor

Info

Publication number: CN114790194B
Application number: CN202011519183.XA
Authority: CN
Inventors: 刘志博; 徐梦欣; 陈俊艺
Original assignee: Suzhou Yaoming Borui Biotechnology Co ltd
Current assignee: Suzhou Yaoming Borui Biotechnology Co ltd
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2024-03-26
Anticipated expiration: 2040-12-21
Also published as: TW202241866A; US20240042066A1; CN114790194A; AU2021408119B2; KR20230118160A; WO2022135326A1; AU2021408119A9; EP4265605A1; AU2021408119A1; JP2024500181A; CA3202738A1

Abstract

The present disclosure provides compounds of formula (I) or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein C is a chelator unit; AB is an albumin binding unit; FAPI is a fibroblast activation protein inhibitor unit. The present disclosure also provides chelates of the above compounds with radionuclides, pharmaceutical compositions, and their use as fibroblast activation protein inhibitors for the diagnosis and treatment of diseases. C-AB-FAPI (I).

Description

Fibroblast activation protein inhibitor

Technical Field

The present disclosure relates to the field of medical and diagnostic, in particular to compounds, chelates, compositions, and uses thereof that inhibit fiber-forming activated proteins.

Background

Tumors are the second largest killer threatening the health of people. Tumors can be considered not only as a collection of malignant cells, but also as a collection of stromal cells, including vascular cells, inflammatory cells, and fibroblasts. In fibroproliferative tumors such as breast, colon and pancreas cancers, the matrix in the tumor may reach 90% or more. One of the tumor stroma is a subpopulation of fibroblasts known as cancer-associated fibroblasts (CAFs) that can be involved in tumor growth, migration and progression and even resist and immunosuppress chemotherapy.

Tumor Microenvironment (TME) around tumors plays an important role in the development of tumors, with activated fibroblasts (CAFs) as the core. Fibroblast activation protein (fibroblast activation protein, FAP) is a type II transmembrane serine proteolytic enzyme belonging to the dipeptidyl peptidase (DPP) family. FAP is selectively expressed on CAFs of more than 90% of epithelial malignant tumors, is hardly expressed in normal tissues, and has special biological characteristics and gene stability. FAP is widely expressed in the microenvironment of a variety of tumors and thus can target different tumor entities, including pancreatic, breast and lung cancers, which account for a significant portion of the solid tumor population. Therefore, the FAP can be used as a biological marker for early diagnosis of tumor, has good targeted therapeutic biological characteristics, and is expected to play an important role in clinical diagnosis and treatment of malignant tumor.

Currently, inhibitors of FAP have not been studied intensively, and the first inhibitor of FAP activity to enter clinical trials was Talabostat, but it shows insufficient clinical activity in various cancers, and thus has not continued further development. Later on, have researcher utilized ¹³¹ I-labeled anti-FAP antibody sibrotuzumab is used for tumor treatment research, but has the defects of low clearance rate, lack of clinical activity and the like.

In recent two years, a series of quinoline-based small molecule-emitting drug-targeted FAP was developed by the university of heidburg, germany, haberkorn, uwe team for diagnosis and treatment, see WO2019154886A1. The resulting inhibitor is able to bind fast and almost completely to FAP in humans and mice, and importantly it does not cross react with DPP family member DPP4, thus laying the foundation for further development. By linking such a FAP inhibitor (FAPI) with the chelating agent DOTA a radionuclide tracer with good pharmacokinetic properties is formed. The most interesting of the whole tracers is FAPI-04, which has a high affinity for FAP, and the tracer is rapidly cleared from the blood and by the kidneys. These characteristics enable ⁶⁸ Tumor imaging of Ga-FAPI-04PET/CT has high contrast and high sensitivity. But rapid elution of FAPI-04 in vivo limits its use in tumor nuclide therapy. Because ofThis is particularly desirable in preserving its excellent targeting and solving the problem of short cycle times for small molecules of FAP inhibitors.

Disclosure of Invention

One aspect of the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt, isomer or solvate thereof,

C-AB-FAPI (I)

wherein C is a chelator unit; AB is an albumin binding unit; FAPI is a fibroblast activation protein inhibitor unit.

In some embodiments, the C unit in formula (I) is selected from:

in some embodiments, the FAPI units in formula (I) are selected from:

in some embodiments, the AB unit in formula (I) comprises a 4-iodo-phenyl end group.

Another aspect of the present disclosure provides a chelate comprising a compound of formula (I) above and a radionuclide.

Yet another aspect of the present disclosure provides a pharmaceutical composition comprising or consisting of: at least one compound of formula (I) above, optionally together with pharmaceutically acceptable excipients.

Yet another aspect of the present disclosure also provides diagnostic or therapeutic uses of the above chelate or pharmaceutical composition, as well as kits.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the following brief description of the drawings of the embodiments will make it apparent that the drawings in the following description relate only to some embodiments of the present disclosure, not to limit the present disclosure.

FIG. 1 illustrates the utilization of ⁶⁸ Ga-TEFAPI-06 imaging assay for healthy miceIn vivo half-life results.

FIG. 2 shows the utilization of ⁶⁸ PET imaging results of Ga-TEFAPI-06 in pancreatic cancer PDX mouse model.

FIG. 3 shows the utilization of ⁸⁶ Long-term PET imaging results of Y-TEFAPI-06 in a pancreatic cancer PDX mouse model.

Fig. 4 shows the results of competition inhibition experiments of TEFAPI-06 in PDX pancreatic cancer mice.

FIG. 5 shows the utilization of ¹⁷⁷ Results of Lu-TEFAPI-06 treatment on PDX pancreatic cancer mice.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present disclosure more apparent, the technical solutions of the embodiments of the present disclosure will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present disclosure. It will be apparent that the described embodiments are some, but not all, of the embodiments of the present disclosure. All other embodiments, which can be made by those skilled in the art based on the described embodiments of the present disclosure without the need for creative efforts, are within the scope of the present invention.

The present invention may be embodied in other specific forms without departing from its essential attributes. It is to be understood that any and all embodiments of the invention may be combined with any other embodiment or features of multiple other embodiments to yield yet further embodiments without conflict. The present invention includes such combinations to yield additional embodiments.

All publications and patents mentioned in this disclosure are incorporated herein by reference in their entirety. If a use or term used in any of the publications and patents incorporated by reference conflicts with a use or term used in the present disclosure, the use or term of the present disclosure controls.

The section headings used herein are for purposes of organizing articles only and should not be construed as limiting the subject matter.

Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning in the art to which the claimed subject matter belongs. In case there are multiple definitions for a term, the definitions herein control.

When any type of range is disclosed or claimed, it is intended that each possible value that the range may reasonably cover be disclosed or claimed separately, including any subrange encompassed therein, unless otherwise indicated. For example, a number of substituents of 1 to 5 indicates an integer within this range, where 1-5 is understood to include 1, 2, 3, 4, 5, and also to include sub-ranges of 1-4 and 1-3.

The description of the present disclosure should be construed as consistent with the principles and principles of chemical bonding. In some cases, it is possible to remove a hydrogen atom in order to accommodate a substituent at a given position.

The use of the terms "comprising," "including," or "containing," and the like, in this disclosure, are intended to cover an element listed after that term as well as equivalents thereof, but do not exclude unrecited elements. The terms "comprising" or "including" as used herein, can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

The term "pharmaceutically acceptable" in this application means: the compound or composition is chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or with the human or mammal with which the disease or condition is to be prevented or treated.

The term "subject" or "patient" includes humans and mammals in this application.

In the context of the present application, the term "treatment" may also include prophylaxis, unless specifically stated to the contrary.

The term "solvate" in this application refers to a complex formed by combining a compound of formula (I) or a pharmaceutically acceptable salt thereof and a solvent. It will be appreciated that any solvate of a compound of formula (I) used in the diagnosis or treatment of a disease or condition described herein, although potentially providing different properties (including pharmacokinetic properties), will result in a compound of formula (I) once absorbed into a subject such that the use of a compound of formula (I) encompasses the use of any solvate of a compound of formula (I), respectively.

The term "hydrate" refers to the case where the solvent in the above term "solvate" is water.

It is further understood that the compound of formula (I) or a pharmaceutically acceptable salt thereof may be isolated in the form of a solvate, and thus any such solvate is included within the scope of the present invention. For example, a compound of formula (I) or a pharmaceutically acceptable salt thereof may exist in unsolvated forms as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like.

The term "pharmaceutically acceptable salt" refers to the relatively non-toxic addition salts of the compounds of the present disclosure. See, for example, S.M. Berge et al, "Pharmaceutical Salts", J.Pharm. Sci.1977,66,1-19.

Suitable pharmaceutically acceptable salts of the compounds of the present disclosure may be acid addition salts of the compounds of the present disclosure having sufficient basicity, e.g., with inorganic acids such as: such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, or acid addition salts with organic acids such as: for example formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectate acid, persulphuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptylic acid, glycerophosphate, aspartic acid, sulfosalicylic acid or thiocyanic acid.

In addition, another suitable pharmaceutically acceptable salt of the compounds of the invention which is sufficiently acidic is an alkali metal salt, such as a sodium or potassium salt, an alkaline earth metal salt, such as a calcium or magnesium salt, an ammonium salt, or a salt with an organic base providing a physiologically acceptable cation, such as a salt with: n-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1, 6-hexamethylenediamine, ethanolamine, glucamine, sarcosine, serinol, tris-hydroxymethyl aminomethane, aminopropanediol, 1-amino-2, 3, 4-butanetriol. Alternatively, the basic nitrogen-containing groups may be quaternized with the following agents: lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and dipentyl sulfate; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides such as benzyl and phenethyl bromides, and the like.

Those skilled in the art will also recognize that the acid addition salts of the claimed compounds can be prepared by any of a variety of known methods by reacting the compounds with an appropriate mineral or organic acid. Alternatively, the alkali metal salts and alkaline earth metal salts of the acidic compounds of the present disclosure are prepared by reacting them with an appropriate base by various known methods.

The present invention includes all possible salts of the compounds of the present disclosure, which may be a single salt or any mixture of the salts in any ratio.

It is to be understood that the term "presently disclosed compounds" as used herein may include, depending on the context: a compound of formula (I), pharmaceutically acceptable salts thereof, solvates of pharmaceutically acceptable salts thereof, and mixtures thereof.

The compounds of the present disclosure may contain one or more asymmetric centers, depending on the desired position and nature of the various substituents. The asymmetric carbon atoms may be present in the (R) or (S) configuration, giving a racemic mixture with one asymmetric center and a diastereomeric mixture with multiple asymmetric centers. In some cases, asymmetry may also be present due to the blocked rotation about a particular bond, for example, the center bond connects two substituted aromatic rings of a particular compound.

Preferred compounds are those that produce a more desirable biological activity. Isolated, purified or partially purified isomers and stereoisomers, or racemic or diastereomeric mixtures of the compounds of the disclosure are included within the scope of the invention. Purification and isolation of such materials can be accomplished by standard techniques known in the art.

The reference herein to "chelator unit" in relation to compounds of formula (I) refers to a fragment of a molecule derived from a chelator. For example, the chelating agent unit is a molecular fragment derived from 1,4,7, 10-tetraazacyclododecane-N, N ', N, N' -tetraacetic acid (DOTA), which may be an amide formed by one carboxyl group of DOTAAnd into the compounds of the general formula (I).

The reference in the present disclosure to a "fibroblast activation protein inhibitor unit" for a compound of formula (I) refers to a molecular fragment derived from a fibroblast activation protein inhibitor. For example, when the inhibitor is a FAPI series of compounds disclosed in Table 1 and Table 3 of WO2019154886A1, the "fibroblast activation protein inhibitor unit" is the removal of R from the FAPI series of compounds ⁸ The resulting molecular fragments.

The "albumin binding unit" mentioned in the present disclosure for the compounds of general formula (I) refers to a molecular fragment having a high affinity to albumin and which has a group attached to a chelator unit and a fibroblast activation protein inhibitor unit.

The reference in the present disclosure to "FAPI units and C units together constitute" for a compound of formula (I) does not refer to direct attachment of the FAPI units and C units in the compound of formula (I), but rather to an artifact situation in which the FAPI units and C units in the compound of formula (I) are pulled out for attachment (removal of the intervening albumin binding unit).

It is to be understood that as used in this disclosure, the singular forms (e.g., "a") can include plural referents unless otherwise specified.

The present disclosure employs standard nomenclature for analytical chemistry, organic synthetic chemistry, and coordination chemistry, and standard laboratory procedures and techniques, unless otherwise indicated. Unless otherwise indicated, the present disclosure employs conventional methods of mass spectrometry, elemental analysis, and the various steps and conditions can be referenced to procedures and conditions conventional in the art.

Reagents and materials used in the present disclosure are commercially available or may be prepared by conventional chemical synthesis methods.

The term "optional" is used herein to describe a situation, and it may or may not occur. For example, the term "optionally substituted" refers to a substituent that is unsubstituted or has at least one non-hydrogen that does not destroy the properties possessed by the unsubstituted analog. For example, the expression "optionally, and pharmaceutically acceptable excipients" as used herein with respect to a pharmaceutical composition means that pharmaceutically acceptable excipients may or may not be present in the pharmaceutical composition.

In the present disclosure, the number of "substitutions" may be one or more, unless otherwise specified; when plural, it may be 2, 3 or 4. In addition, when the number of "substitutions" is plural, the "substitutions" may be the same or different.

In the present disclosure, the "substituted" position may be any position unless otherwise specified.

The term "C" as used herein ₁ -C ₁₀ Alkyl "means a straight or branched alkane chain containing from 1 to 10 carbon atoms. For example, C ₁ -C ₆ Representative examples of alkyl groups include, but are not limited to, methyl (C ₁ ) Ethyl (C) ₂ ) N-propyl (C) ₃ ) Isopropyl (C) ₃ ) N-butyl (C) ₄ ) Tert-butyl (C) ₄ ) Sec-butyl (C) ₄ ) Isobutyl (C) ₄ ) N-pentyl (C) ₅ ) 3-pentanyl (C) ₅ ) Neopentyl (C) ₅ ) 3-methyl-2-butanoyl (C) ₅ ) Tert-amyl (C) ₅ ) And n-hexyl (C) ₆ ) Etc. The term "lower alkyl" isRefers to straight or branched alkyl groups having 1 to 4 carbon atoms. "substituted alkyl" refers to an alkyl group substituted at any available point of attachment with one or more substituents, preferably 1 to 4 substituents. The term "haloalkyl" refers to an alkyl group having one or more halogen substituents including, but not limited to, e.g., -CH ₂ Br、-CH ₂ I、-CH ₂ Cl、-CH ₂ F、-CHF ₂ -CF ₃ Such groups.

The term "alkylene" as used herein refers to a divalent hydrocarbon radical as described above for "alkyl" but having two points of attachment. For example, methylene is-CH ₂ -group, ethylene is-CH ₂ -CH ₂ -a group.

The terms "alkoxy" and "alkylthio" as used herein refer to an alkyl group as described above that is attached via an oxygen linkage (-O-) or a sulfur linkage (-S-), respectively. The terms "substituted alkoxy" and "substituted alkylthio" refer to a substituted alkyl group attached via an oxygen or sulfur bond, respectively. "lower alkoxy" is a group OR, where R is lower alkyl (alkyl containing 1 to 4 carbon atoms).

The term "halogen" as used herein refers to fluorine, chlorine, iodine or bromine.

Albumin has been increasingly used as a drug carrier, and is often used to improve the hemodynamic properties of drugs, thereby increasing the half-life of the blood stream. Albumin is the most abundant protein in human plasma and is responsible for various storage and transport tasks in the body. Compared with normal tissue, the tumor tissue has rich blood vessels and larger vascular endothelial gaps, albumin can permeate into the tumor tissue as a macromolecular substance and cannot enter the normal tissue, in addition, substances with smaller molecular weight are cleared from the tumor interstitium more quickly, and macromolecules are trapped, so that the effect is also called a permeability enhancement and retention effect (enhanced permeability and retention effect, EPR) of the macromolecular substance in the tumor tissue. In addition, tumor microenvironments have high expression of albumin-binding receptors, such as gp60 receptor and SPARC134, which further retain albumin near the tumor. Thus, the use of albumin as a carrier for anticancer drugs not only improves the half-life of these drugs, but also improves delivery to and retention in the tumor. The albumin carrier system mainly comprises albumin carriers which are chemically coupled and physically combined.

The present disclosure links albumin binding agents with chelator units and FAP inhibitor units, thereby forming small molecule compounds (TEFAPI) that can be dual-targeted with FAP and albumin, with the aim of extending the blood circulation half-life of the FAPI molecule and increasing tumor uptake.

The present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt, isomer or solvate thereof,

C-AB-FAPI (I)

In one embodiment, the C unit is derived from a chelating agent selected from the group consisting of: 1,4,7, 10-tetraazacyclododecane-N, N ', N, N ' -tetraacetic acid (DOTA), ethylenediamine tetraacetic acid (EDTA), 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N, N, N ', N ', N ' -pentaacetic acid (DTPA), bis- (carboxymethyl imidazole) glycine or 6-hydrazinopyridine-3-carboxylic acid (HYNIC).

For example, the C unit isIt is derived from 1,4,7, 10-tetraazacyclododecane-N, N ', N, N' -tetraacetic acid (DOTA), which can form an amide +.>And into the compounds of the general formula (I).

For example, the C unit is It is derived from 11,4,7-triazacyclononane-1, 4, 7-triacetic acid (NOTA), which can form an amide +.>Is introduced into the general formulaI) Is a compound of (a).

In one embodiment, the C unit in the compound of formula (I) is selected from:

note that it is possible to make a simple solution,through the terminal carbonyl group of which is attached to the AB unit, i.e. can also be expressed asSimilarly, a->Through its terminal carbonyl group to the AB unit, can also be represented asSimilarly, a->Is linked to the AB unit via its terminal carbonyl group, i.e.can also be denoted as +.>

In one embodiment, the FAPI unit in the compound of formula (I) is selected from

In one embodiment, the FAPI units and C units in the compound of formula (I) satisfy the following conditions:

if the FAPI unit and the C unit are linked (with the intermediate albumin binding unit removed), the new compound resulting from the linkage is selected from the group consisting of:

in other words, in this embodiment, the compound of formula (I) may be regarded as having an albumin binding unit AB inserted into the molecular structure of the compounds FAPI-02, FAPI-04, FAPI-21, FAPI-34, FAPI-42, FAPI-46, FAPI-52, FAPI-69, FAPI-70, FAPI-71, FAPI-72, FAPI-73, FAPI-74. The compounds FAPI-02, FAPI-04, FAPI-21, FAPI-34, FAPI-42, FAPI-46, FAPI-52, FAPI-69, FAPI-70, FAPI-71, FAPI-72, FAPI-73, FAPI-74 are disclosed in WO2019154886A1 as an FAP inhibitor.

In a preferred embodiment, the FAPI units and C units in the compounds of formula (I) meet the following conditions: if the FAPI unit and the C unit are linked (with the intermediate albumin binding unit removed), the new compound resulting from the linkage is selected from the group consisting of: FAPI-04, FAPI-21 or FAPI-46. In one embodiment, the FAPI units and C units in the compound of formula (I) satisfy the following conditions: if the FAPI unit and the C unit are linked (with the intermediate albumin binding unit removed), the new compound obtained by the ligation is FAPI-04.

In one embodiment, the AB unit comprises a 4-iodo-phenyl end group.

In one embodiment, the AB unit is through a terminus in the FAPI unit An amide bond is formed to connect it to the FAPI unit and the AB unit connects it to the C unit by forming an amide bond with the terminal carbonyl group in the C unit.

In one embodiment, the compounds of formula (I) are

Or a pharmaceutically acceptable salt, isomer or solvate thereof.

Based on the Jansen et al design of small molecule FAP inhibitors (FAPI) with high affinity, loktev et al first developed radiotracers FAPI-01 and FAPI-02, which can rapidly bind to and internalize FAP in human and murine cells. The accumulation in normal tissue is very small and the clearance speed is fast, so that a high contrast can be obtained for PET imaging. In addition, FAPI-02 can be cleared rapidly from the organism by renal clearance without remaining in the renal parenchyma, which is advantageous for therapeutic applications. To optimize uptake and tracer retention in tumors, a series of FAPI-02 based compounds were developed, with PET imaging of FAPI-04 showing higher tumor uptake, longer residence time, and no significant increase in activity in normal organs. FAPI-04 has been used in clinical experiments to image PET of 28 patients with different cancers, and found that only the cancer sites are ingested while the normal tissues are hardly ingested, and shows very excellent cancer targeting properties, and the diagnostic reagent has been used in clinic. Because the FAP target is an excellent therapeutic target, for patients with metastatic advanced stage, conventional methods such as surgery, radiotherapy and chemotherapy and the like cannot inhibit tumor development, the life of the patients is prolonged, and the FAP inhibitor is a promising treatment method for carrying the radionuclide. It is therefore desirable to address the short cycle time of FAP inhibitor small molecules while retaining their excellent targeting properties.

TEFAPI-06 is a structure of 4- (p-iodophenyl) butyric acid derivative introduced into FAPI-04. The introduction of the iodobenzene structure with high affinity with albumin in the FAPI-04 structure prolongs the in-vivo blood circulation time of a Fibroblast Activation Protein (FAP) inhibitor, can be specifically and highly ingested at a tumor part, and after PET imaging tests, the imaging metabolic rule of the inhibitor shows that the blood half-life is prolonged from 26 minutes to 400 minutes. In lutetium-177 radiotherapy experiments, the tumor of mice was significantly inhibited. TEFAPI-06 is expected to find application in imaging and radionuclide carriers for a variety of cancers.

The present disclosure also provides a chelate comprising:

a compound of the above formula (I) or a pharmaceutically acceptable salt, isomer or solvate thereof, and

a radionuclide.

In the chelate, the chelator unit is directly chelated with the radionuclide (e.g., ⁶⁸ chelation of Ga with chelating agent units derived from DOTA), or indirect incorporation of radionuclides by chelation with other metals (e.g., al ³⁺ Chelation with a chelating agent unit derived from DOTA, a radionuclide ¹⁸ F is introduced into the chelate in the form of a counterion).

In one embodiment, the radionuclide is selected from: ¹⁸ F、 ⁵¹ Cr、 ⁶⁷ Ga、 ⁶⁸ Ga、 ¹¹¹ In、 ⁹⁹ mTc、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ¹³⁹ La、 ¹⁴⁰ La、 ¹⁷⁵ Yb、 ¹⁵ 3Sm、 ¹⁶⁶ Ho、 ⁸⁶ Y、 ⁸⁸ Y、 ⁹⁰ Y、 ¹⁴⁹ Pm、 ¹⁶⁵ Dy、 ¹⁶⁹ Er、 ¹⁷⁷ Lu、 ⁴⁷ Sc、 ¹⁴² Pr、 ¹⁵⁹ Gd、 ²¹² Bi、 ²¹³ Bi、 ⁷² As、 ⁷² Se、 ⁹⁷ Ru、 ¹⁰⁹ Pd、 ¹⁰⁵ Rh、 ^101m Rh、 ¹¹⁹ Sb、 ¹²⁸ Ba、 ¹²³ I、 ¹²⁴ I、 ¹³¹ I、 ¹⁹⁷ Hg、 ²¹¹ At、 ¹⁵¹ Eu、 ¹⁵³ Eu、 ¹⁶⁹ Eu、 ²⁰¹ Tl、 ²⁰³ Pb、 ²¹² Pb、 ⁶⁴ Cu、 ⁶⁷ Cu、 ¹⁸⁸ Re、 ¹⁸⁶ Re、 ¹⁹⁸ Au、 ²²⁵ Ac、 ²²⁷ Th and ¹⁹⁹ ag. For example, the radionuclide is ⁶⁸ Ga、 ⁸⁶ Y or ¹⁷⁷ Lu。

The present disclosure also provides a pharmaceutical composition comprising or consisting of:

at least one of the above-mentioned chelate compounds,

optionally, and pharmaceutically acceptable excipients.

In one embodiment, the pharmaceutical composition comprises or consists of at least one of the chelates described above. In another embodiment, the pharmaceutical composition comprises or consists of at least one of the chelates described above and a pharmaceutically acceptable excipient.

The compositions of the present disclosure may also, as necessary or desired, comprise pharmaceutically acceptable excipients that formulate the chelate for the intended route of administration. Adjuvants include, but are not limited to, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, coating materials, and the like. Adjuvants are generally described in "Remington's Pharmaceutical Sciences" of e.w. martin. Examples of excipients include, but are not limited to, aluminum monostearate, aluminum stearate, carboxymethyl cellulose, sodium carboxymethyl cellulose, crospovidone, glycerol isostearate, glycerol monostearate, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxyoctacosanol stearate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose monohydrate, magnesium stearate, mannitol, microcrystalline cellulose, and the like.

Agents that may be used to formulate the compositions for the intended route of administration include:

acidulants (examples include, but are not limited to, acetic acid, citric acid, fumaric acid, hydrochloric acid, nitric acid);

alkalizing agents (examples include, but are not limited to, aqueous ammonia, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide, triethanolamine;

buffers (examples include, but are not limited to, potassium metaphosphate, dipotassium phosphate, sodium acetate, sodium citrate anhydrous and sodium citrate dihydrate); etc.

WO2019154886A1 discloses that chelates or compositions comprising a FAPI series compound and a radionuclide can be used for diagnosing or treating a disease characterized by overexpression of fibroblast activation protein in a mammal or human. The present disclosure retains its excellent FAP targeting by introducing albumin binding units into the structure of the FAPI compounds disclosed in WO2019154886A1, while extending the circulation time of FAP inhibitors.

Another aspect of the present disclosure relates to the use of the chelate or composition described above for the diagnosis or treatment of a disease characterized by overexpression of fibroblast activation protein in a mammal or human. For example, a disease characterized by overexpression of Fibroblast Activation Protein (FAP) is selected from the group consisting of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, and scar disease, preferably wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian nest cancer, hepatocellular cancer, esophageal cancer, hypopharynx cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocellular carcinoma, clear cell renal cancer, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, CUP (primary unknown cancer), thymus cancer, glioma, astrocytoma, cervical cancer, and prostate cancer.

Yet another aspect of the present disclosure relates to a kit comprising or consisting of the chelate complex described above or the pharmaceutical composition described above, and instructions for diagnosing or treating a disease. In a preferred embodiment, the disease is the above-described disease characterized by overexpression of fibroblast activation protein.

Examples

The starting materials for the examples are commercially available and/or may be prepared by a variety of methods well known to those skilled in the art of organic synthesis. Those skilled in the art of organic synthesis will appropriately select the reaction conditions (including solvents, reaction atmospheres, reaction temperatures, duration of the experiment, and post-treatments) in the synthetic methods described below. Those skilled in the art of organic synthesis will appreciate that the functional groups present on each part of the molecule should be compatible with the reagents and reactions presented.

The following abbreviations are respectively indicated:

1. TEFAPI-06 chemical Synthesis

All reagents and compounds synthesized are commercially available in China (without Kong Australian platform), and suppliers include national pharmaceutical group chemical reagent Limited, saen chemical technology (Shanghai) Limited, jiudng chemical (Shanghai) technology Limited, beijing carboline technology Limited, beijing Tong Guangxi chemical Co., shanghai Pico pharmaceutical technology Limited, beijing Yinuoki technology Limited, shanghai Michelia Biochemical Co., sigma Aldrich (Shanghai) trade Limited. The key starting materials are all marked with CAS numbers on the circuit diagram.

TEFAPI-06 was synthesized by the above chemical route. The building block can be decomposed into 5 parts of building block 4 synthesis, building block 12 synthesis, building block 13 synthesis, building block 20 synthesis and building block connection, and certain prepositive requirements are met.

And (3) building block 4 synthesis:

a) To a solution of compound 5978-22-3 (20 g,53.64mmol,1eq, HCl) in methylene chloride (200 mL) was added tetrahydropyran-2, 6-dione (9.18 g,80.45mmol,1.5 eq) and then the solution was cooled to 0℃followed by dropwise addition of triethylamine (16.28 g,160.91mmol,22.40mL,3 eq) followed by stirring at 20℃for 1 hour. After completion of the reaction, the mixture was concentrated to give compound 1 (20 g, crude product) as a colorless oil. MS (ESI) ⁺ ):m/z 451.2(M+H) ⁺

b) To a solution of Compound 1 (20 g,44.39mmol,1 eq) in methanol (200 mL) was added Pd/C (8 g,10% purity), followed by H ₂ The reaction was carried out in an atmosphere of (15 psi) for 12 hours. After the completion of the reaction, the mixture was concentrated under reduced pressure to give compound 2 (14 g, crude product) as a colorless oily substance. MS (ESI) ⁺ ):m/z 317.2(M+H) ⁺

c) To a solution of compound 27913-58-2 (15 g,51.71mmol,1 eq) in methylene chloride (150 mL) at 0deg.C were added 1-hydroxypyrrolidine-2, 5-dione (6.55 g,56.88mmol,1.1 eq) and dicyclohexylcarbodiimide (12.80 g,62.05mmol,12.55mL,1.2 eq) followed by a reaction at room temperature for 1 hour. After the reaction is finished, filtering and activating to obtain filtrate, and concentrating under reduced pressure to obtain pale yellow solid The body was then dissolved in dimethylformamide (120 mL). And compound 2 (12 g,37.93mmol,1 eq) and N, N-diisopropylethylamine (14.71g,113.78mmol,19.82mL,3 eq) were added, followed by reaction at room temperature for 2 hours. The reaction mixture was concentrated and purified by prep-HPLC to give compound 3 (10 g,16.57mmol,43.68% yield, 97.5% purity) as a colorless oily liquid. MS (ESI) ⁺ ):m/z 533.1(M/2+H) ⁺

d) To a solution of Trt-Cl resin (3.85 g,76.3mmol,1 eq) in dichloromethane (500 mL) was added compound 71989-26-9 (42.90 g,91.56mmol,1.2 eq), N-diisopropylethylamine (29.58 g,228.90mmol, 39.87mL,3 eq), stirred at room temperature, and nitrogen sparged for 48 hours. Subsequently, the mixture was sequentially treated with dichloromethane (5×500 mL), CH ₃ OH (5 x 500 ml) and dimethylformamide (5 x 500 ml). A piperidine/dimethylformamide (v/v=1:5,400 ml) solution was then added and nitrogen sparged for 20 minutes followed by a washings with dimethylformamide (5 x 300 ml). Dimethylformamide (300 mL) was then added and the reaction was cooled to 0deg.C, and compound 3 (18.14g,30.43 mmol,1.1eq), 6-chlorobenzotriazole-1, 3-tetramethyluronium hexafluorophosphate HCTU (22.84g,56.96 mmol,2eq), 1-hydroxybenzotriazole HOBT (1.92 g,14.14mmol,0.5 eq) and N, N-diisopropylethylamine (10.8 g,86.44mmol,14.9mL,3 eq) were added in that order followed by a return to room temperature for 2 hours. Followed by washing with dimethylformamide (4 x 400 mL) and dichloromethane (4 x 400 mL), then dichloromethane/trifluoroacetic acid (v/v=100:1, 600 mL) was added and the reaction was bubbled for 20 minutes and filtered. NaHCO was used as filtrate ₃ (aq) was neutralized and extracted with dichloromethane (200 ml x 3) and water (200 ml x 3) in portions, the combined organic phases were washed with citric acid (200 ml x 3) and subsequently evaporated under reduced pressure to give compound 4 (6 g,7.11mmol,74.99% yield, 96.77% purity) as a yellow solid. ¹ H NMR(400MHz,CDCl ₃ )δ＝1.30-1.40(m,2H),1.43-1.51(m,24H),1.91-1.93(m,6H), 2.16-2.21(m,7H),2.56-2.60(m,3H),3.07-3.11(m,3H),3.47-3.49(m,1H),4.48-4.50(m,1H),4.57-4.60(m,1H),4.76-4.79(m,1H),5.86(br s,1H),6.90-6.94(m,2H),7.58-7.61(m, 2H),7.88-7.91(m,1H),8.09-8.12(m,1H)。MS(ESI ⁺ ):m/z 817.4(M+H) ⁺

The building block 12 is synthesized:

a) NH was slowly added to a solution of compound 203866-15-3 (10 g,37.70mmol,1 eq) in MeOH (25 mL) over 20 min at 0deg.C ₃ (28.89 g,1.70mol,45 eq) in MeOH (125 mL). The temperature was then returned to room temperature at N ₂ The reaction was stirred for 18 hours under an atmosphere of (15 psi). After the reaction was completed, it was concentrated by evaporation under reduced pressure, and slurried with PE/MTBE to give compound 8 (8.9 g,35.57mmol,94.34% yield) as a yellow solid. ¹ H NMR(400MHz DMSO)：δ1.28-1.45(m,9H),2.16-2.38(m,1H),2.59-2.87(m,1H),3.55-3.84(m,2H),4.08-4.36(m,1H),6.97-7.19(m,1H),7.36-7.61(m,1H)

b) Pyridine (3.38 g,42.68mmol,3.44mL,1.2 eq) was slowly added to a solution of compound 8 (8.9 g,35.57mmol,1 eq) in dichloromethane (100 mL) over 20 min at 0deg.C. Trifluoroacetic anhydride (8.96 g,42.68mmol,5.94ml,1.2 eq) was added while maintaining 0 ℃, followed by reaction was brought to room temperature and stirred for 18 hours, followed by concentration under reduced pressure. Beating using PE/MTBE gave compound 9 (7.5 g,32.30mmol,90.81% yield) as a yellow solid. ¹ H NMR (400 MHz chloroform-d): δ1.33-1.66 (m, 9H), 2.71 (br t, j=9.04 hz, 2H), 3.80 (br s, 2H), 4.52-4.90 (m, 1H)

c) To a solution of compound 9 (7.0 g,30.14mmol,1 eq) in acetonitrile (200 mL) was added HCl/dioxane (4 m,14.00mL,1.86 eq) followed by reaction for 18 hours. The solid was filtered and washed with MTBE (100 mL) to give compound 10 (2.2 g,13.05mmol,43.30% yield, HCl) as a white solid. ¹ H NMR(400MHz DMSO)δ2.64-3.13(m,2H),3.48-3.89(m,2H),4.99(br t,J＝6.95Hz,1H),9.80(br s,2H)

d) To 10mL of dimethylformamide were added, in order, 2- (tert-butoxycarbonylamino) acetic acid (779.41 mg, 4.45mmol,1.5 eq), 1-hydroxybenzotriazole HOBT (80.16 mg, 593.22. Mu. Mol,0.2 eq), 2- (IH-benzotriazol-1-yl) -N, N, N ', N' -tetramethylisourea phosphorus hexafluoride HBTU (2.25 g,5.93mmol,2 eq) and compound 10 (500 mg,2.97mmol,1eq, HCl), followed by stirring of the reaction for 30 minutes. N, N-diisopropylethylamine (1.15 g,8.90mmol,1.55mL,3 eq) was then added and stirred for 16 hours. After the reaction is finished, water is added(20 mL) and extracted with ethyl acetate (50 mL), and the organic phase was concentrated and purified using silica gel column chromatography (PE: ethyl acetate=1:1) to give compound 11 (0.245 g,2.13mmol,71.68% yield). ¹ H NMR(400MHz DMSO):δ1.38(m, 9H),2.78(m,2H),3.77(m,2H),4.06(m,1H),4.23(m,1H),5.07(d,J＝5.2Hz,1H),7.14(m, 1H)

e) To a solution of compound 11 (0.4 g,1.38mmol,1 eq) in acetonitrile (13.5 mL) at 0 degrees celsius HCl/dioxane (4 m,2.67mL,7.71 eq) was added, followed by reaction back to room temperature for 16 hours. Concentration gave compound 12 (322 mg,1.28mmol,92.89% yield, 90% purity, HCl) as an off-white solid. ¹ H NMR(400MHz DMSO):δ2.81-3.01(m,2H),3.57(s,2H),3.69(br d,J＝5.01Hz,1H), 3.78-3.89(m,1H),3.91-4.14(m,2H),4.24(ddd,J＝15.59,11.41,4.11Hz,1H),5.19(dd, J＝8.70,3.34Hz,1H),8.38(br s,3H)

And (3) building block 13 synthesis:

a) Compound 137076-54-1 (150 mg, 261.90. Mu. Mol,1 eq) was dissolved in acetonitrile (15 mL), and 1-hydroxypyrrolidine-2, 5-dione (33.16 mg, 288.09. Mu. Mol,1.1 eq), HBTU (109.26 mg, 288.09. Mu. Mol,1.1 eq) were added in this order, followed by stirring for 12 hours, and after drying the mixture under reduced pressure, it was purified using prep-TLC (EA: CAN=7:1) to give compound 13 (115 mg, 171.69. Mu. Mol,65.56% yield) as a pale yellow solid, which was used directly in the next step.

And (3) building block 20 synthesis:

a) Compound 52351-75-4 (20 g,112.89mmol,1 eq) and KOH (69.68 g,1.24mol,11 eq) were dissolved in H ₂ To O (200 mL) was then added pyruvic acid (10.94 g,124.18mmol,8.75mL,1.1 eq) and the reaction mixture was reacted at 40℃for 15 hours. The reaction was then cooled to 15 ℃ and acidified to pH 3 with hydrochloric acid, the solid was filtered and washed with deionized water to give compound 14 (24 g,92.23mmol,81.70% yield, 9) as an off-white solid5% purity) ¹ H NMR(400MHz DMSO):δ3.70-4.24(m,3H),7.58(dd,J＝9.24,2.80 Hz,1H),8.15(d,J＝9.30Hz,1H),8.26(d,J＝2.74Hz,1H),8.53(s,1H),13.75(br s,2H),MS(ESI+):m/z 248.1(M+H) ⁺

b) To compound 14 (24 g,97.09mmol,1 eq) was added nitrobenzene (150 mL) and stirred at 220 ℃ for 1.5 hours. The reaction was then cooled to 15 ℃ and PE (150 mL) was added and the precipitated solid was washed with PE (100 mL) to give compound 15 (16.8 g,82.68mmol,85.16% yield) as an off-white solid. ¹ H NMR(400MHz DMSO)δ3.72-4.14(m,3H),7.49(dd,J＝9.23,2.75Hz,1H),7.93(d,J＝4.40Hz,1H),8.02(d,J＝9.17Hz,1H),8.18(d,J＝2.69Hz,1H),8.87(d,J＝4.40Hz,1H),13.76(br s,1H)。 MS(ESI+):m/z 202.1(M-H) ^-

c) To compound 15 (40 g,196.86mmol,1 eq) was added hydrogen bromide (1L, 48% in water) and stirred at 130℃for 12 hours. Subsequently, the reaction was basified to ph=6 using 350ml of 30% sodium hydroxide solution, a large amount of precipitate was precipitated, the precipitate was filtered, and the precipitate was washed with methanol and dried to give a crude product, and washed with methanol multiple times to give the product as brown solid compound 16 (30 g,128.93mmol,65.50% yield, 81.3% purity). ¹ H NMR(400MHz,DMSO-d ₆ )δppm 13.66(br s,1H),10.24(s,1H),8.77(d,J＝4.5Hz,1H), 8.06(d,J＝2.6Hz,1H),7.95(d,J＝9.1Hz,1H),7.84(d,J＝4.4Hz,1H),7.36(dd,J＝2.7, 9.1Hz,1H)。MS(ESI ^- ):m/z 377.0(2M-H) ^-

d) To a solution of compound 16 (30 g,158.59mmol,1 eq) in dimethylformamide (300 mL) were added potassium carbonate (87.67 g,634.36mmol,4 eq) and 1-bromo-3-chloro-propane (24.97 g,158.59mmol,15.60mL,1 eq), followed by stirring at 60 ℃ for 12 hours. After concentration by evaporation, 200mL of water was added to the system, a large amount of solids precipitated, and the reaction mixture was stirred at 25 ℃ for 15 minutes and suction filtered. Trituration using ethyl acetate and filtration gave compound 17 as a brown solid (37.58 g,130.13mmol,86.15% yield, 92% purity). ¹ H NMR(400 MHz,DMSO-d ₆ )δppm 2.26-2.98(m,2H),3.83-3.88(m,2H),4.22-4.25(m,2H), 7.54(dd,J＝9.19,2.69Hz,1H),7.96(d,J＝4.38Hz,1H),8.03-8.11(m,1H),8.19(d,J＝2.63Hz,1H),8.90(d,J＝4.38Hz,1H)。MS(ESI ⁺ ):m/z 266.1(M+H) ⁺ ,

e) Compound 17 (30 g,112.91mmol,1 eq) was added to N-methylpyrrolidone (300 mL) and tert-butyl piperazine-1-carboxylate (105.15 g,564.56mmol,5 eq) and potassium iodide (9.37g,56.46mmol,0.5 eq) were added followed by stirring at 60℃for 12 hours. The reaction mixture was cooled to room temperature and filtered, and the crude product was purified by Prep-HPLC (0.1% FA) to give compound 18 (42.96 g,96.16mmol,85.16% yield, 93% purity) as a yellow solid. ¹ H NMR(400MHz,DMSO-d ₆ )δppm 8.82(d,J＝4.4Hz,1H),8.19-8.16(m,1H),7.99(d,J＝ 9.3Hz,1H),7.85(d,J＝4.4Hz,1H),7.45(dd,J＝2.8,9.1Hz,1H),4.15(br t,J＝6.2Hz,2H),2.62-2.50(m,6H),2.42(br t,J＝4.6Hz,4H),1.99(br d,J＝6.6Hz,2H),1.39(s, 9H)。MS(ESI ⁺ ):m/z 416.1(M+H) ⁺

f) To a solution of compound 18 (12.00 g,26.57mmol,92% purity, 1.02 eq) in dimethylformamide (120 mL) was added O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate (19.76 g,52.10mmol,2 eq), 1-hydroxybenzotriazole (7.04 g,52.10mmol,2 eq), N-diisopropylethylamine (10.10 g,78.15mmol, 13.61mL,3 eq), followed by compound 12 (5.88 g,26.05mmol,1.00eq, HCl) and the reaction was stirred at 25℃for 12 hours. Filtration and purification of the filtrate using prep-HPLC (HCl) gave compound 19 (11.27 g,17.29mmol,66.37% yield, 90% purity) as a yellow solid. ¹ H NMR(400MHz,DMSO-d ₆ )δppm 1.41(s,9 H),2.27-2.33(m,2H),3.27-3.29(m,5H),3.43-3.55(m,2H),3.93-4.21(m,10H), 5.19(dd,J＝9.26,2.75Hz,1H),7.59(dd,J＝9.26,2.63Hz,1H),7.69(d,J＝4.63Hz,1H)7.87-8.00(m,1H),8.14(d,J＝9.26Hz,1H),8.95(d,J＝4.63Hz,1H),9.24(br t,J＝ 5.82Hz,1H),11.00(br s,1H).。MS(ESI ⁺ ):m/z 587.2(M+H) ⁺ ,

g) Compound 19 (2 g,3.41mmol,1 eq) was dissolved in ethyl acetate (40 mL) and a hydrochloric acid/ethyl acetate solution (4M, 4mL,4.69 eq) was added followed by reaction at 25℃for 6 hours. The reaction mixture was concentrated by evaporation and used directly to give compound 20 (1.5 g, crude, HCl) as a yellow solid. MS (ESI) ^- ):m/z 485.1(M+H) ⁺

And (3) building block connection:

a) Compound 4 (858.97 mg,1.05mmol,1.1 eq) was dissolved in dimethylformamide (5 mL), HBTU (453.23 mg,1.20mmol,1.25 eq), HOBT ((167.94 mg,1.24mmol,1.3 eq), N-diisopropylethylamine (617.83 mg,4.78mmol,832.65uL,5 eq) and compound 20 (229.65mg,333.04 umol,1eq) were added and the mixture was stirred for 12 hours. After concentration of the reaction product, purification using prep-HPLC (HCl) afforded compound 21 (230 mg,170.00umol,17.78% yield, 95% purity) as a white solid. MS (ESI) ⁺ ):m/z 1285.4(M+H) ⁺

b) Compound 21 (150 mg,116.71 mol,1 eq) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (3.08 g,27.01mmol,2mL,231.45 eq) was added and the reaction stirred for 4 hours followed by concentration by evaporation to give the crude product as a yellow oily compound 22 (150 mg, crude, trifluoroacetic acid). MS (ESI) ⁺ ):m/z 1129.3(M+H) ⁺

c) Compound 22 (150 mg,120.67umol,1eq, trifluoroacetic acid) was dissolved in dimethylformamide (1.6 mL), N-diisopropylethylamine (124.77 mg,965.36umol,168.15ul,8 eq) and compound 13 (88.91 mg,132.74umol,1.1 eq) were added in this order, the reaction was stirred for 12h, and then the solvent was concentrated in vacuo. The crude product, yellow oily compound 23 (220 mg, crude product) was obtained. MS (ESI) ⁺ ):m/z 1684.9(M/2+H) ⁺

d) Compound 23 (210 mg,124.72 mol,1 eq) was added to trifluoroacetic acid (3.23g,28.36mmol,2.10 mL,227.41eq) and stirred for 3 hours. Followed by concentration under reduced pressure and purification using prep-HPLC (trifluoroacetic acid) gave compound 24 (TEFAPI-06) (30 mg, 26.40. Mu. Mol,21.16% yield, 100% purity) as a white solid.

The white solid compound 24 (TEFAPI-06) is characterized as follows:

LCMS(ESI ⁺ ):m/z 758.4 1/2(M+2H) ⁺ Rt:1.959min.

LC conditions: kinetex C18.times.2.1 mm column (5 μm particle size) 1.0ml/min

Gradient: phase a 0.05% trifluoroacetic acid/water, phase B0.05% trifluoroacetic acid/acetonitrile. 0-0.40min 5% B,0.40-3.00 min 5-95% B,3.00-4.00min 95% B.

HPLC:Rt:2.266min.

LC conditions: luna-C18 2.0 x 50mm column (5 μm particle size), phase a 0.05% trifluoroacetic acid/water, phase B0.05% trifluoroacetic acid/acetonitrile.

LC gradient: 0.00-4.90min:10-80% B0.8 mL/min,4.90-5.50min:0% B1.2 mL/min.

¹ H NMR(400MHz,DMSO-d ₆ )

δppm 9.18-9.07(m,1H),8.84(d,J＝4.4Hz,1H),8.49(br s,1H),8.18-7.97(m,3H), 7.89(br d,J＝2.4Hz,1H),7.78(br t,J＝5.8Hz,1H),7.62(br d,J＝8.1Hz,2H),7.55(d,J＝ 4.4Hz,1H),7.46(br dd,J＝2.7Hz,9.1Hz,1H),7.00(br d,J＝8.1Hz,2H),5.15(br dd,J＝3.3, 9.0Hz,1H),4.69(br s,1H),4.47-4.08(m,6H),4.07-3.69(m,13H),3.64-3.22(m,11H),3.21-2.69(m,14H),2.47(br s,2H),2.33-1.90(m,9H),1.87-1.18(m,18H).

¹⁹ F NMR(400MHz,DMSO-d ₆ )-73.867

¹ H NMR (400 MHz, methanol-d) ₄ )

δppm 8.85(d,J＝4.6Hz,1H),8.22-7.97(m,2H),7.71-7.53(m,4H),6.99(d,J＝8.3Hz, 2H),5.15(br d,J＝9.3Hz,1H),4.83-4.74(m,4H),4.49-4.30(m,6H),4.30-4.08(m,4H),3.90-3.38(m,17H),3.20-2.74(m,16H),2.58(s,2H),2.40(br s,2H),2.30-2.15(m,6H),2.02 -1.82(m,6H),1.77-0.99(m,11H).

¹⁸ F NMR (400 MHz, methanol-d) ₄ )-77.161

2. By means of ⁶⁸ In vivo half-life determination by Ga-TEFAPI-06 imaging on healthy mice

The gallium germanium generator was rinsed with 5mL of 0.6M high purity hydrochloric acid to give Ga-68 hydrochloric acid solution. 1mL of the rinsed Ga-68 solution was taken, 100. Mu.L of 3M sodium hydroxide and 130. Mu.L of 3M sodium acetate were added to adjust the acidity to a final pH of 4.0, 50. Mu.g of TEFAPI-06 precursor was added, and the reaction mixture was heated to 90℃for 10 minutes. The reaction solution was passed through a C18 column, free ions were removed, and then the C18 column was eluted with an ethanol solution. Obtaining marked ⁶⁸ Ga-TEFAPI-06。

Taking 37MBq marked product, adding 200 microliters of normal saline for dilution, and extracting the medicine by an insulin syringe for standby, wherein the ethanol content of the medicine is not higher than 5%. Healthy mice were previously anesthetized and placed on a PET/CT acquisition bed with an indwelling needle placed in the tail vein. The syringe is connected to the indwelling needle, and the acquisition of PET data is started at the zero time of pushing the needle. The collection time points are respectively 0-60 minutes, 2 hours, 3 hours, 4 hours and 5 hours. And after the acquisition is finished, reconstructing the data by using professional software under the condition that the data are reconstructed every minute at intervals of the first 5 minutes, reconstructing the data every 5 minutes for 5-60 minutes, and reconstructing the data once at the rest time points. The acquired data are processed by PET reconstruction software to obtain continuous images. In PET processing software, fixed areas were delineated at the heart of the mice, resulting in SUV-Mean and SUV-Max values at the heart. The resulting SUV was simulated in data processing software. The corresponding blood half-life was obtained, giving a half-life of TEFAPI-06 of 398 minutes, as shown in FIG. 1.

3. ⁶⁸ PET imaging of Ga-TEFAPI-06 in pancreatic cancer PDX mouse model

The gallium germanium generator was rinsed with 5mL of 0.6M high purity hydrochloric acid to give Ga-68 hydrochloric acid solution. 1mL of the rinsed Ga-68 solution was taken, 100. Mu.L of 3M sodium hydroxide and 130. Mu.L of 3M sodium acetate were added to adjust the acidity to a final pH of 4.0, 50. Mu.g of TEFAPI-06 precursor was added, and the reaction mixture was heated to 90℃for 10 minutes. The reaction solution was passed through a C18 column, free ions were removed, and then the C18 column was eluted with an ethanol solution. Obtaining marked ⁶⁸ Ga-TEFAPI-06. Will be marked ⁶⁸ Ga-TEFAPI-06 was diluted with physiological saline and 3.7MBq was injected into each mouse. PET scan imaging was performed at 0.5 hours, 1.5 hours, 2.5 hours, and reconstruction was performed using PET image processing software. The obtained image is shown in FIG. 2, and the probe can be seen to have obvious uptake at the tumor site.

4. ⁸⁶ Long-term PET imaging of Y-TEFAPI-06 in pancreatic cancer PDX mouse model

Y-86 is a positron species having a half-life of up to 14.6 hours and can be radiolabeled with DOTA and is therefore well suited for long-term TE detectionDistribution of FAPI molecules in vivo. 1mL of Y-86 hydrochloric acid solution was taken, 100. Mu.L of 3M sodium hydroxide and 130. Mu.L of 3M sodium acetate were added to adjust the acidity to a final pH of 4.0, 50. Mu.g of TEFAPI-06 precursor was added, and the reaction mixture was heated to 90℃for 10 minutes. The reaction solution was passed through a C18 column, free ions were removed, and then the C18 column was eluted with an ethanol solution. Obtaining marked ⁸⁶ Y-TEFAPI-06. Will be marked ⁸⁶ Y-TEFAPI-06 was diluted with physiological saline and each mouse was injected with 7.4MBq. PET scan imaging at 0.5, 2, 6, 12, 18, 24, 36 hours after injection, respectively, was reconstructed using PET image processing software. The resulting PET images are shown in fig. 3, with SUV values plotted at the tumor and heart of the mice, and the change in SUV values over time is plotted. Tumor uptake its SUV uptake profile indicates that tumor uptake was continuously rising for the first 24 hours, blood circulation time was longer, and was continuously cleared from the kidneys.

5. Competitive inhibition assay of TEFAPI-06 in PDX pancreatic cancer mice

To verify the specificity of TEFAPI-06 for FAP targets, we performed PET imaging of the same batch of mice before and after inhibition in a competitive inhibition experiment. From the long-term PET imaging of Y-86, it is known that the uptake of tumor peaks between 18 hours and 24 hours after drug injection. Thus, the tumor-bearing mice are firstly subjected to ⁶⁸ Ga-FAPI-04 imaging, mice were determined to have tumor uptake. Waiting for 48 hours to ensure ⁶⁸ After Ga-FAPI-04 metabolism, 300. Mu.g of TEFAPI-06 molecules were injected into these two mice, and after 18 hours the mice injected with TEFAPI-06 molecules were subjected to ⁶⁸ PET molecular imaging of Ga-FAPI-04. After injection ⁶⁸ PET imaging results of Ga-FAPI-04 for 30 minutes showed significant uptake at tumors without TEFAPI-06 injection, and no additional uptake at mice tumors after TEFAPI-06 injection.

As shown in fig. 4, there was substantially no additional uptake at the tumor site in the mice undergoing inhibition, whereas the uptake was higher at the tumor site in the same batch of mice without inhibition. The results indicate that the targeting site of TEFAPI-06 is FAP.

6. ¹⁷⁷ Lu-TEFAPI-06 treatment of PDX pancreatic cancer mice

Tumor targeting and mouse metabolic regulation based on TEFAPI-06, the excellent tumor/normal tissue uptake ratio is greater than 10 after 18 hours, and kidney metabolism is basically cleared, so that the PDX pancreatic cancer mice are subjected to Lu-177 radioactive targeting treatment. 5 mice per group were treated as shown in the following graph with a 3.7MBq dose and a tumor growth curve that can be seen to completely suppress the tumors in the treated group. To evaluate the effect of treatment, we performed an imaging physical examination of FAPI-04 on mice before and after treatment, and the results are shown in FIG. 5. It can be seen that after treatment, the uptake of the prototumor in the mice had substantially disappeared.

The foregoing is merely exemplary embodiments of the present invention and is not intended to limit the scope of the invention, which is defined by the appended claims.

Claims

1. Compounds of formula (I)Or a pharmaceutically acceptable salt, isomer thereof.

2. A chelate complex comprising:

(1) A compound of claim 1, or a pharmaceutically acceptable salt, isomer thereof; and

(2) A radionuclide.

3. The chelate of claim 2, wherein the radionuclide is selected from the group consisting of: ¹⁸ F、 ⁵¹ Cr、 ⁶⁷ Ga、 ⁶⁸ Ga、 ¹¹¹ In、 ⁹⁹ mTc、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ¹³⁹ La、 ¹⁴⁰ La、 ¹⁷⁵ Yb、 ¹⁵ 3Sm、 ¹⁶⁶ Ho、 ⁸⁶ Y、 ⁸⁸ Y、 ⁹⁰ Y、 ¹⁴⁹ Pm、 ¹⁶⁵ Dy、 ¹⁶⁹ Er、 ¹⁷⁷ Lu、 ⁴⁷ Sc、 ¹⁴² Pr、 ¹⁵⁹ Gd、 ²¹² Bi、 ²¹³ Bi、 ⁷² As、 ⁷² Se、 ⁹⁷ Ru、 ¹⁰⁹ Pd、 ¹⁰⁵ Rh、 ^101m Rh、 ¹¹⁹ Sb、 ¹²⁸ Ba、 ¹²³ I、 ¹²⁴ I、 ¹³¹ I、 ¹⁹⁷ Hg、 ²¹¹ At、 ¹⁵¹ Eu、 ¹⁵³ Eu、 ¹⁶⁹ Eu、 ²⁰¹ Tl、 ²⁰³ Pb、 ²¹² Pb、 ⁶⁴ Cu、 ⁶⁷ Cu、 ¹⁸⁸ Re、 ¹⁸⁶ Re、 ¹⁹⁸ Au、 ²²⁵ Ac、 ²²⁷ th and ¹⁹⁹ Ag。

4. the chelate of claim 3 wherein the radionuclide is ⁶⁸ Ga、 ⁸⁶ Y or ¹⁷⁷ Lu。

5. A pharmaceutical composition comprising or consisting of:

at least one chelate according to any one of claims 2 to 4,

optionally, and pharmaceutically acceptable excipients.

6. Use of a chelate according to any one of claims 2-4 or a pharmaceutical composition according to claim 5 in the manufacture of a reagent for diagnosing or treating a disease characterized by overexpression of Fibroblast Activation Protein (FAP) in a subject.

7. The use according to claim 6, wherein the disease characterized by overexpression of Fibroblast Activation Protein (FAP) is selected from the group consisting of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and scar disease.

8. The use of claim 7, wherein the cancer is selected from breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular cancer, esophageal cancer, hypopharynx cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocellular carcinoma, clear cell renal cancer, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, CUP (primary unknown cancer), thymus cancer, glioma, astrocytoma, cervical cancer, and prostate cancer.

9. The use of claim 7, wherein the cancer is selected from gliomas.

10. A kit comprising or consisting of a chelate according to any one of claims 2-4 or a pharmaceutical composition according to claim 5, and instructions for diagnosing or treating a disease.